ALBERT

Description: Background: In this study we cloned a vector based on a self-inactivating lentiviral backbone containing MDR1 (multidrug resistance 1 gene) connected by an ECMV-IRES-element with MGMT P140K (O6-BG-resistant mutant of O6-methylguanine-DNA methyltransferase). The chemoprotective potential of this HR′SIN-MDR-IRES-MGMT combination vector was compared to single vectors (HR′SIN-MDR, HR′SIN-MGMT). Methods: HL60 and CD34+ cells were transduced with the various vectors. After chemotherapeutical treatment MTT assays were used to detect chemoresistance levels in HL60 cells, CD34+ cells were held in liquid culture under differentiation conditions and analysed by FACS for MDR1 expression. Results: HL60 cells transduced with the combination vector showed significant chemoresistance to O6-BG/ACNU (IC50 13x higher compared to untransduced control), the IC50 of cells transduced with HR′SIN-MGMT was 35x higher. The IC50 of paclitaxel (MDR1 substrate) was 24x higher in cells transduced with HR’SIN-MDR and 25x higher with HR’SIN-MDR-IRES-MGMT compared to untransduced control. Combined exposure of cells to O6-BG/ACNU and paclitaxel showed a survival advantage of cells transduced with the combination vector (IC50 6.25x higher), for the single vectors the IC50 was 1.63x higher (MDR1) and 2.08x higher (MGMT) compared to untransduced control. Treatment of CD34+ cells with increasing concentrations of doxorubicin (up to 0.8 μM) resulted in a higher fraction of MDR1-positive cells either with HR′SIN-MDR (26.6x) or with HR′SIN-MDR-IRES-MGMT (30.6x) compared to untreated cells. After combination treatment (20μM O6-BG/16μM BCNU and 0.4μM doxorubicin) the fraction of MDR1-positive cells was higher for HR′SIN-MDR-IRES-MGMT (14x) than HR′SIN-MDR (8x) transduced cells. Conclusion: The protective effect of the combination vector is comparable with the single vectors for monotherapy and superior for combined therapy. The combination vector presents simultaneous protective effects of two drug resistance genes, thus reducing the risk of insertional mutagenesis by one transduction process. Consequently our results might help to reduce myelotoxic side effects and increase the chemotherapeutic efficiency.

Description: Background: High dose chemotherapy with autologous stem cell transplantation (ASCT) improves disease free and overall survival in pts with MM; tandem ASCT may further enhance clinical benefits. Mobilization with G-CSF alone fails to yield sufficient CD34+ cells for a tandem ASCT in the majority of MM pts. A mobilization regimen including G-CSF and cyclophosphamide is more effective, but associated with significant toxicity. This phase II study evaluated the efficacy and safety of a non-cytotoxic mobilization regimen of AMD3100 plus G-CSF for ASC mobilization in pts with MM. Methods: Mobilization treatment consisted of subcutaneous G-CSF (Filgrastim 10 μg/kg) given in the morning on 5 consecutive days and a single dose of AMD3100 (240 μg/kg) in the evening of day 4, 10–11 hours prior to leukapheresis. These procedures could be repeated for up to 5 additional days in order to collect an adequate number of cells for transplantation. Monitoring of CD34+ cells in peripheral blood (PB) was performed immediately prior to each AMD3100 administration and prior to the aphereses. Patients were treated with high dose chemotherapy in preparation for transplantation according to local standard of care guidelines. Pts did not receive hematopoietic growth factors following ASCT. The primary endpoint of the study was safety; secondary endpoints included 1) % of pts with 〉2-fold increase of CD34+ cells following AMD3100, and 2) % of pts in with hematopoietic recovery between day 14 and day 21. Results: 31 pts were evaluable, including 19 males (median age: 57 yrs, range: 40–73) and 12 females (median age: 61 yrs, range: 53–67). Pts had received a maximum of 4 prior chemotherapy cycles. The cumulative proportion of pts reaching a target of 5×10^6/kg CD34+ cells was 60% on day 1, 87% on day 2, and 93% on day 3. For the initial mobilization, AMD3100 increased absolute CD34+ counts ≥ 2-fold in 78% of pts (median fold increase: 2.8; range: 1.1–15.2). Additional aphereses showed a ≥ 2-fold increase in 21% of cases (median fold increase: 1.4; range: 0.7–6.5). Overall, the median number of CD34+ cells yielded was 7.1×10^6/kg (range: 3–28×10^6/kg). The majority of pts (n=19) underwent only a single apheresis, whereas additional procedures were needed in 12 cases. Nineteen pts received a single transplant, 11 pts a tandem transplant. Autografts contained a median of 3.1×10^6/kg CD34+ cells (range 2.4–9.2×10^6/kg). After first transplant, median time to neutrophil and platelet engraftment was 14 and 13 days, respectively. All patients had complete engraftment within 20 days except 1 pt who had neutrophil recovery at day 34 (single transplant) and 1 had platelet recovery at day 27 after the 2nd transplant. AMD3100 was well tolerated, drug-related adverse events (AEs) were limited to 2 cases of mild nausea/vomiting. Conclusions: The addition of AMD3100 to G-CSF doubled the number of mobilized CD34+ cells in the majority of pts, allowing the collection of sufficient CD34+ cells for tandem ASCT in 1–2 aphereses. Unlike chemotherapeutics commonly used to enhance stem cell mobilization, AMD3100 was not associated with any significant toxicity.

Description: Gene transfer of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) into hematopoietic stem cells has been shown to protect hematopoiesis from the toxic side effects of O6-guanine alkylating drugs such as BCNU, ACNU or temozolomide (TMZ). In addition, MGMT gene transfer allows efficient in vivo selection of transduced hematopoietic stem cells and enrichment of genetically corrected cells in the context of gene therapy for monogenetic diseases. We here have analysed the long-term effect of MGMT gene transfer on the hematopoietic stem cell compartment using an in vivo murine transplantation/gene therapy model and a retroviral vector carrying the gene for MGMTP140K, a mutant resistant to the wtMGMT-specific inhibitor O6-benzylguanine (BG). Serial transplants were performed and primary, secondary as well as tertiary recipients were treated with combined BG/ACNU, BG/BCNU or BG/TMZ chemotherapy at doses myeloablative in non-MGMT-protected hematopoiesis. Serial transplantation was performed with 1.8 – 3.0 x 106 mononuclear bone marrow cells and 2 to 3 animals were transplanted per primary or secondary animal. While initial gene transfer efficiency was low (1–5% of cells engrafted at week four) chemotherapy resulted in efficient selection of transduced cells in primary animals (70–90% transgene expression in peripheral blood). Secondary and tertiary recipients showed 40–80% transgene expression even before CTX. Efficient stem cell engraftment and protection from CTX was demonstrated in 〉 90% of secondary animals, while tertiary recipients clearly demonstrated compromised engraftment and a substantial number of animals did not survive CTX treatment. Retroviral vector integration site analysis to study the clonality of hematopoiesis of stem cells by ligation mediated PCR (LM-PCR) was performed in the serially transplanted mice. In three mice of the secondary transplantation cohort we detected 3, 0, and 6 clones, respectively. In three mice of the tertiary transplantation cohort 7, 2, and 2 clones, respectively, were found. Thus, an exhaustion of transduced hematopoiesis following regenerative stress by high dose chemotherapy was not evident. Of the total 20 detected clones one could not be mapped to the mouse genome, while the others could be blasted against the mouse genome (assembly 2004, http://genome.ucsc.edu/; 〉99.5% identity). It turned out that 5 of 8 integrations landed in RefSeq in the tertiary transplantation cohort, while 3 of 8 integrations occurred in RefSeq genes in the secondary transplantation cohort. This suggests that clones profit from the transcription machinery of their integration site. Thus, our LM-PCR results indicate that the multiclonality of hematopoiesis is conserved after serial transplants which may be considered a safety feature for drug-resistance gene therapy. Furthermore, vector integration in highly resistant stem cells is favored in actively transcribed genomic regions.

Description: Insertional mutagenesis and development of leukemia following retroviral gene therapy has created intense interest in assessing the safety of viral vectors for further gene therapy trials. Using the gtsg.org database we analyzed more than 14,900 different viral integration sites of ASLV, FIV, FV, HIV, MLV and SIV based vectors in terms of insertions into fragile sites, cancer genes, transcription factor binding sites, CpG islands, and repetitive elements (SINE, LINE, LTR elements). When we compared these data with our newly generated random set, containing 1,000,000 random integrations, we discovered that the gene density on fragile sites strongly correlates to the HIV vector insertion frequency. Furthermore, we report a up to a five fold increased frequency of HIV, MLV and SIV insertions in cancer genes. The majority of cancer genes preferentially hit by HIV viruses were found associated to acute leukemias, while MLV and SIV vector insertion sites are seen more evenly spread over the cancer gene repertoire. When analyzing different cell entities, it turned out that CD34+ hematopoetic stem cells had highest rates of intragenic insertions and hosted significantly more HIV and FV insertions in cancer genes than other cell types, such as HeLa, T cells, 293T cells, macrophages, fibroblasts, or SupT1 cells.

Description: It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral multidrug-resistance 1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analysis on the clonal level were lacking. One method to study the in vivo behaviour of repopulating progenitor and stem cells is marking the cells with replication-incompetent retroviral vectors that integrate into identifiable host DNA sequences, thus allowing the tracking of cell progeny based on unique proviral insertion sites. In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were split into two aliquots and transduced either with a multidrug-resistance 1 gene-retroviral vector based on the Harvey murine sarcoma virus (HaMDR1-vector) or a NeoR-retroviral vector based on the Moloney murine leukaemia virus (G1Na-vector). After autologous retransplantation, DNA from blood and bone marrow was collected at different time points in a period of 4 years and the animals are still alive. By using a highly sensitive and specific ligation-mediated polymerase chain reaction (LM-PCR) followed by sequencing of vector integration sites, we found in animal M120 32 different contributing hematopoietic clones 8 weeks and 50 weeks after transplantation and in animal M038 17 clones 58 weeks after transplantation. Based on the difference between the sequences of the HaMDR1-LTR and the G1Na-LTR, the clones can be allocated definitely to one of the two vectors. Remarkably, 36 clones descend from the G1Na-vector, whereas only 13 clones descend from the HaMDR1-vector. We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

Description: Introduction: It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral MDR1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analyses on the clonal level and a long-term follow-up in a model with more direct relevance to human HSC biology were lacking. Methods: In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were transduced either with a MDR1 gene-retroviral vector (HaMDR1-vector) or a NeoR-retroviral vector (G1Na-vector). After autologous transplantation granulocytes and mononuclear cells of each animal were obtained at different time points and analysed by using a highly sensitive and specific ligation-mediated PCR (LM-PCR) and by quantitative real time PCR. Results: In monkey M120 73 different cell populations (clones) were detected between 8 weeks and 4 years after transplantation; in monkey M038 49 clones could be identified between 16 weeks and 4 years after transplantation. Remarkably, 99 clones descend from the G1Na-vector, whereas only 23 clones descend from the HaMDR1-vector. Quantitative real time PCR analyses support these data: in both animals, the mean proportion of the G1Na-vector and the HaMDR1-vector, respectively, is 2,46%±0,014% and 0,455%±0,003%. Furthermore, single G1Na clones identified with LM-PCR 4 years after transplantation were quantified, showing a copy number between 1.43±0,52 and 7.1±0.34, according to 0.001%±0.0003% and 0.004%±0.0003% of the granulocytes fraction. In the MNC fraction, the G1Na clones were not detectable. At the same time point, 2 HaMDR1 clones were quantified, one in the granulocyte and one in the MNC fraction, respectively. Here, the copy numbers were 2.87±0.56 and 3.52±1.11, according to 0.002%±0.0003% and 0.002%±0.0008% of the granulocyte and MNC fraction, respectively. Conclusions: We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

Description: Resistance of chronic myelogenous leukemia (CML) to imatinib is a serious clinical challenge. In vivo targeted gene therapy may be a strategy to overcome resistance. For gene therapy of CML standard rAAV2-based vectors lack the required gene transfer efficiency as well as the in vivo selectivity. An advancement in vector development (Muller et al., Nat. Biotech., 2003) now allows the generation of rAAV capsid mutants that offer higher target cell specificity and efficiency. We used the “AAV random peptide library” method to generate capsid mutants efficient and specific for CML cell lines, primary CML cells and primary human peripheral blood progenitor cells PBPC. After previously applying this method successfully on CML cell lines, an effective vector for CML cell lines could be obtained. On primary human CML and PBPC the new vector was more efficient than rAAV2 but still at an unsatisfying level. To increase the gene transfer on primary cells we applied the “AAV library” on primary human CML and PBPC. A total of 44 different mutant capsid clones were obtained, most of them at three or less copies. Four clones were observed 〉10x to 39x and were selected for further testing. To determine efficiency and specificity, a panel of leukemic (CML, imatinib-sensitive and -resistant, and AML) and other solid tumor cell lines were transduced (moi 100). On a panel of leukemic cell lines similar or higher gene transfer efficiency of the rAAV capsid mutants was observed compared to standard rAAV2 vectors. The higher transduction efficiency in imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S was particularly striking, as the difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S (table 1). On solid tumor cell lines rAAV2 was more efficient, confirming the higher specificity of our capsid mutants. Compared to our previous work (

Description: Chronic myelogenous leukemia (CML) has gained outstanding importance for targeted cancer therapy. Inhibition of the BCR-ABL tyrosine kinase by imatinib (STI571, Glivec, Gleevec) leads to reduced proliferation of CML cells in vitro and sustained hematological and cytogenetic responses in vivo. However, resistance has been observed after variable periods of imatinib monotherapy especially in advanced stages of disease and overexpression of the BCR-ABL protein is one of the mechanisms of imatinib resistance. As combination therapy may allow to overcome drug resistance, we were interested in the effect of combination treatment with imatinib and 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the heat shock protein 90 (Hsp90) chaperone complex. Furthermore, a new mechanism of action of the heat Hsp90 inhibitor 17-AAG is brought to light giving implications for an additional benefit of a combination treatment of imatinb-resistant chronic myelogenous leukemia. In imatinib-sensitive CML cell lines, combination index values (CI) obtained using the method of Chou and Talalay indicated additive (CI = 1) or slightly antagonistic (CI 〉 1) effects following simultaneous treatment with imatinib and 17-AAG. In contrast, the agents acted synergistically in imatinib-resistant BCR-ABL overexpressing LAMA84-R cells (CI = 0.6 at 75 % growth inhibition level). Growth inhibition of CFU-GM colonies of primary CML cells obtained from 3 patients is stronger after combination treatment than after monotherapy and annexin V / propidium iodide staining showed a strong increase of the apoptotic cell fraction in CML cells treated for 48 hrs by the combination treatment as compared to treatment with each drug alone. In imatinib-resistant cells BCR-ABL mRNA levels and protein expression were increased compared to the imatinib-sensitive parental cell line which is consistent to our cytogenetic and multicolor FISH analyses revealing multiple genomic BCR-ABL copies in imatinib-resistant cells. Furthermore, in imatinib-resistant cells P-glycoprotein activity was increased. Single treatment with 17-AAG lowered BCR-ABL and increased Hsp70 protein levels in both cell lines as expected whereas combination treatment was even more effective in this respect. Interestingly, single treatment with 17-AAG also decreased P-glycoprotein activity in a dose-dependent fashion as confirmed by a rhodamine-123 exclusion assay. The synergistic effect of both drugs in imatinib-resistant cells may thus be explained by increased intracellular levels of imatinib following 17-AAG treatment. The relevance of this additional mechanism warrants further exploration in clinical studies.

Description: Analysis of the fate of retrovirally transduced cells after transplantation is often hampered by the scarcity of available DNA. We evaluated a promising method for whole genome amplification named multiple displacement amplification (MDA) with respect to the even and accurate representation of retrovirally transduced genomic DNA. We were able to show that MDA is a suitable method to subsequently specify engraftment efficiencies by quantitative real-time PCR as the retroviral integrations are amplified the same way and by the same probability as all other parts of the genome. We validated the method by analyzing a dilution series containing retrovirally transduced DNA and untransduced background DNA and retroviral integrations found in primary material from a retroviral transplantation model by quantitative real-time PCR. The representation of the portion of retroviral DNA in the amplified samples was 0.9-fold (range 0.2 – 2.1-fold) of the portion determined in the original genomic DNA. Furthermore, the succession of the combination of MDA and integration site analysis by ligation-mediated PCR showed an increase in the sensitivity of the method as a specific integration site could be detected in a background of untransduced DNA, while the transduced DNA made up only 0.001%. These results show that MDA enables large scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow up analysis in gene therapy studies even from smallest amounts of starting material.

Description: Normal haematopoietic stem cells are particularly sensitive to radiation-induced apoptosis. Overexpression of the multidrug resistance 1 (MDR1) gene product, P-glycoprotein (P-gp), leads to suppression of apoptosis and is radioprotective in the human lymphoblastoid cell line TK6. Therefore, gene therapy with MDR1 might protect bone marrow cells during tumour-radiotherapy. The aim of this study was to test if human CD34(+) blood stem cells can be protected from the effect of radiation by MDR1 gene transduction. MDR1 cDNA was cloned into the lentiviral SIN-vector pHR’SINcPPT-SEW to replace the eGFP cDNA. CD34(+) cells isolated from four individual donors were exposed to lentiviral supernatants with an MOI of 10 of either HR’SIN-MDR1 or HR’SINcPPT-SEW as a control. After lentiviral transduction, 0.8×105 cells of transduced and non-transduced CD34(+) control cells, respectively, were irradiated with 0–8 Gy and held in liquid culture under differentiation conditions. Ten days after irradiation, the amount of MDR1 expressing cells was determined by the Rhodamine-(Rh−)123 efflux assay and the MDR1-expression rate was monitored by real-time PCR. Additionally, the differentiation status was tested with FACS-analyses for CD11b (myeloid and natural killer cells), CD15 (neutrophils, eosinophils, monocytes), CD33 (myeloid progenitor cells, monocytes), and CD34 (hematopoietic precursors) expression. To test the potential of MDR1 to protect differentiated cells, unirradiated cells were irradiated after 10 days in liquid culture. Apoptotic cells were detected 48 hours later by staining with Annexin V. The transduction efficiency for the MDR1-virus was 3–18%, for the GFP-virus 12–60%. The proportion of RH-123-negative (=MDR1-positive) cells of all four donors increased with escalating radiation doses (e.g. 18–54% from 0–8 Gy). Irradiation of the differentiated cells after ten days of liquid culture also led to an increase of RH-123-negative cells with escalating radiation doses (e.g. 12.5–17% from 0–8 Gy). We found a correlation between radiation dose and differentiation status: independent on transduction the amount of CD11b-cells increased at 2–4 Gy (e.g from 23–45%) and decreased to 9% with 8 Gy; a similar course was also seen for CD15 expression. Our results clearly indicate the radioprotective effect of human blood stem cells by lentiviral MDR1-overexpression. Thus, enhancing repopulation by surviving stem cells may increase the irradiation tolerance of hematopoietic cells and thus contribute to widening the therapeutic range in radiotherapy.